Recombinant DNA technology has enabled the expression of heterologous proteins in bacteria and other host cells at relatively high levels. Often, heterologous protein produced by the host cell precipitates inside the cell as opposed to being soluble in the cell. Methods to isolate these refractile bodies containing heterologous proteins offer substantial advantages to industrial scale purification schemes.
One such purification scheme is the invention disclosed in commonly owned U.S. Pat. No. 4,748,234, which is incorporated herein by reference in its entirety. It describes a process for recovering refractile bodies containing a heterologous protein from a host cell by first disrupting the cell wall and cell membrane of the microorganism. At least 99% of the salts are then removed from the disruptate and the desalted disruptate is redisrupted. Subsequently, the viscosity of the liquid is increased and the refractile bodies are separated from the cellular debris by high speed centrifugation. This process is effective, however, there is always a need for improvements which reduce time, labor or materials. Accordingly, it was discovered that if the first two steps are reversed, the third step can be eliminated. That is, if salt removal precedes cell disruption, the second disruption is unnecessary. Additionally, if the disruption is sufficient to result in a monolayer pellet rather than a bilayer pellet after centrifugation, the addition of a material to increase the density or viscosity of the disruptate is unnecessary. This invention, therefore, involves improvements in the methods disclosed in U.S. Pat. No. 4,748,234 whereby the number of processing steps is reduced, processing time is decreased, the process is easily scaled-up and maximum recovery of refractile material containing heterologous expression products is the ultimate result.
Becker et al., Biotech. Advs. (1983) 1:247-261 disclose the separation of refractile bodies from most of the cell debris and soluble impurities by a low-speed centrifugation. In addition, Kleid et al., ch. 25 in Developments in Industrial Microbiology, Vol. 25, p. 317-235 (Society for Industrial Microbiology, Arlington, Va. (1984) disclose purification of refractile bodies by homogenization followed by centrifugation. Also, Marston et al., Bio/Technology (September, 1984), pp. 800-804 describe release of inclusion bodies by enzymatic and mechanical disruption procedures as well as sonication. Purification and activity assurance of precipitated heterologous proteins is also described by U.S. Pat. Nos. 4,511,502; 4,511,503; 4,512,922; 4,518,526 and 4,620,948; and EP 114,506. These disclosures indicate that numerous steps involving repeated cell disruptions, resuspensions and centrifugations were required to obtain an isolated refractile body preparation. PCT application WO 85/05637 discloses a purification for gamma interferon which forms refractile bodies in E. coli involving cell disruption and subsequent centrifugation. U.S. Pat. No. 4,677,196, EP application 218,374 and EP application 212,960 describe the extraction, purification and activation of proteins produced as insoluble inclusion bodies. Insoluble material is obtained by disruption and centrifugation. U.S. Pat. No. 4,656,255 and EP application 215,625 discloses a process for increasing yields of recombinant proteins by recovering sidestream precipitates after initial solubilization and chromatography of inclusion bodies.
There remains a need in the art for a method of recovering refractile material containing heterologous expression products from the host cells which method is less costly, is easier to handle, faster and results in maximum recovery of pure protein in a biologically active form without use of chemical agents.